Diet formulations

This application is a U.S. National Stage Application under 35 U.S.C. § 371 of International Patent Application No. PCT/US2020/065892, filed Dec. 18, 2020, which claims priority to U.S. Provisional Patent Application No. 62/950,747, filed Dec. 19, 2019 and U.S. Provisional Patent Application No. 63/075,972 filed Sep. 9, 2020, the disclosure of each are incorporated by reference in their entirety.

Not applicable.

Not applicable.

The sequence listing provided in the file named 20191217_NB41781-US-PSP_Sequence Listing_ST25 with a size of 130 KB which was created on Dec. 17, 2019 and which is filed herewith, is incorporated by reference herein in its entirety.

Not applicable.

Phytase is the most commonly used exogenous enzyme in feed for monogastric animals. Phytase can reduce the antinutritional effect of phytate and improve the digestibility of phosphorous, calcium, amino acids and energy, as well as reduce the negative impact of inorganic phosphorous excretion to the environment.

Phytate is the major storage form of phosphorus in cereals and legumes. However, monogastric animals such as pig, poultry and fish are not able to efficiently metabolize or absorb phytate (or phytic acid) in their diet and therefore it is excreted, leading to phosphorous pollution in areas of intense livestock production. Moreover, phytic acid also acts as an anti-nutritional agent in monogastric animals by chelating metal agents such as calcium, copper and zinc and forming insoluble complexes with proteins and amino acids in various segments of the digestive tract.

Through the action of exogenous phytase, phytate is generally hydrolysed to give lower inositol-phosphates and inorganic phosphate. Phytases are useful as additives to animal feeds where they improve the availability of organic phosphorus to the animal and decrease phosphate pollution of the environment (Wodzinski R J, Ullah A H. Adv Appl Microbiol. 42, 263-302 (1996)). Nevertheless, despite the action of supplied phytases in animal feed, in order to provide sufficient phosphates for growth and health of these animals, inorganic phosphorus must still be added to their diets, especially in young broilers from 0-10 days of age. Such addition can be costly and further increases pollution problems.

Accordingly, a need exists for the development of animal diets, particularly for poultry, that are substantially or completely inorganic phosphate free, while still ensuring normal growth characteristics in all growth phases compared to phytase-containing diets that are supplemented with sources of inorganic phosphate such as monocalcium phosphate and/or meat and bone meal.

The field pertains to poultry diets containing no or substantially no or decreased inorganic phosphate and/or no meat bone meal as well as engineered phytase polypeptides and uses of the same for enhancing animal performance.

Provided herein, inter alia, are animal diets containing phytases which are free or substantially free of exogenously added inorganic phosphate or contain substantially reduced exogenously added inorganic phosphate. When fed to poultry, these diets ensure normal growth during all phases of development compared to diets which contain inorganic phosphate supplementation.

Accordingly, in one aspect, provided herein is a poultry diet comprising (i) an engineered phytase polypeptide or a fragment thereof comprising phytase activity; and (ii) no or substantially no inorganic phosphorus. In some embodiments, said phytase polypeptide or a fragment thereof comprising phytase activity comprises at least 82% sequence identity with the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, said phytase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID: NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37. In some embodiments of any of the embodiments disclosed herein, the diet further comprises one or more of (a) a direct fed microbial comprising at least one bacterial strain, (b) at least one other enzyme, and/or (c) an essential oil. In some embodiments of any of the embodiments disclosed herein, the engineered phytase polypeptide or fragment thereof is present in an amount of at least about 0.1 g/ton feed. In some embodiments of any of the embodiments disclosed herein, the diet further comprises calcium from about 0.62 to 0.72% in a starter diet, about 0.54 to 0.64% in a grower diet, and/or about 0.42 to 0.55% in a finisher diet. In some embodiments, the finisher diet comprises from about 0.46 to 0.55% calcium. In other embodiments, the finisher diet comprises from about 0.42 to 0.50% calcium. In some embodiments of any of the embodiments disclosed herein, the diet comprises amino acids (e.g., digestible lysine) from about 1.18 to 1.22% in a starter diet, about 1.06 to 1.10% in a grower diet, and/or about 0.88 to 1.0% in a finisher diet. In some embodiments, the finisher diet comprises from about 0.96 to 1.0% amino acids (e.g., digestible lysine). In other embodiments, the finisher diet comprises from about 0.88 to 0.92% amino acids (e.g., digestible lysine). In some embodiments of any of the embodiments disclosed herein, the diet comprises metabolizable energy from about 2824 to 2950 kcal/kg in a starter diet, about 2924 to 3050 kcal/kg in a grower diet, and/or about 2970 to 3120 kcal/kg in a finisher diet. In some embodiments, the finisher diet comprises from about 2970 to 3100 kcal/kg metabolizable energy. In other embodiments, the finisher diet comprises from about 2994 to 3120 kcal/kg metabolizable energy. In some embodiments of any of the embodiments disclosed herein, the diet comprises sodium from about 0.13 to 0.17% for all phases (starter, grower, and finisher diets). In some embodiments of any of the embodiments disclosed herein, the diet contains a phytate source comprising one or more of corn, wheat, soybean meal, rapeseed, rice and/or wheat bran. In some embodiments of any of the embodiments disclosed herein, the diet further comprises oat hulls. In some embodiments of any of the embodiments disclosed herein, the diet lacks meat and/or bone meal. In some embodiments of any of the embodiments disclosed herein, the diet further comprises one or more additional feed enzymes selected from the group consisting of a xylanase, a protease, an amylase, and a glucoamylase. In some embodiments of any of the embodiments disclosed herein, the diet is a starter diet. In some embodiments of any of the embodiments disclosed herein, the diet is a grower diet. In some embodiments of any of the embodiments disclosed herein, the diet is a finisher diet. In some embodiments of any of the embodiments disclosed herein, the poultry is a broiler or a layer.

In additional aspects, provided herein is a method for improving animal performance on one or more metrics comprising administering an effective amount of any of the diets disclosed herein to the animal. In some embodiments, the one or more metrics is selected from the group consisting of increased feed efficiency, increased weight gain, reduced feed conversion ratio, improved digestibility of nutrients or energy in a feed, improved nitrogen retention, improved ability to avoid the negative effects of necrotic enteritis, and improved immune response. In some embodiments of any of the embodiments disclosed herein, the animal is poultry. In some embodiments of any of the embodiments disclosed herein, the poultry is selected from the group consisting of turkeys, ducks, chickens, geese, pheasants, quail, and emus. In some embodiments, the chicken is selected from the group consisting of layers and broilers. In some embodiments, egg production in layers improves by up to 12.5% compared to egg production in layers that are fed an inorganic-phosphate free diet that is not supplemented by any of the phytases disclosed herein. In other embodiments, egg mass improves by up to 15.6% compared to egg mass from layers that are fed an inorganic-phosphate free diet that is not supplemented by any of the phytases disclosed herein.

The following sequences comply with 37 C.F.R. §§ 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and the sequence listing requirements of the European Patent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administrative Instructions. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. § 1.822.

SEQ ID NO:1 corresponds to the predicted mature sequence of engineered phytase PHY-13594.

SEQ ID NO:2 corresponds to the predicted mature sequence of engineered phytase PHY-10931.

SEQ ID NO:3 corresponds to the predicted mature sequence of engineered phytase PHY-10957.

SEQ ID NO:4 corresponds to the predicted mature sequence of engineered phytase PHY-11569.

SEQ ID NO:5 corresponds to the predicted mature sequence of engineered phytase PHY-11658.

SEQ ID NO:6 corresponds to the predicted mature sequence of engineered phytase PHY-11673.

SEQ ID NO:7 corresponds to the predicted mature sequence of engineered phytase PHY-11680.

SEQ ID NO:8 corresponds to the predicted mature sequence of engineered phytase PHY-11895.

SEQ ID NO:9 corresponds to the predicted mature sequence of engineered phytase PHY-11932.

SEQ ID NO:10 corresponds to the predicted mature sequence of engineered phytase PHY-12058.

SEQ ID NO:11 corresponds to the predicted mature sequence of engineered phytase PHY-12663.

SEQ ID NO:12 corresponds to the predicted mature sequence of engineered phytase PHY-12784.

SEQ ID NO:13 corresponds to the predicted mature sequence of engineered phytase PHY-13177.

SEQ ID NO:14 corresponds to the predicted mature sequence of engineered phytase PHY-13371

SEQ ID NO:15 corresponds to the predicted mature sequence of engineered phytase PHY-13460.

SEQ ID NO:16 corresponds to the predicted mature sequence of engineered phytase PHY-13513.

SEQ ID NO:17 corresponds to the predicted mature sequence of engineered phytase PHY-13637.

SEQ ID NO:18 corresponds to the predicted mature sequence of engineered phytase PHY-13705.

SEQ ID NO:19 corresponds to the predicted mature sequence of engineered phytase PHY-13713.

SEQ ID NO:20 corresponds to the predicted mature sequence of engineered phytase PHY-13747.

SEQ ID NO:21 corresponds to the predicted mature sequence of engineered phytase PHY-13779.

SEQ ID NO:22 corresponds to the predicted mature sequence of engineered phytase PHY-13789.

SEQ ID NO:23 corresponds to the predicted mature sequence of engineered phytase PHY-13798.

SEQ ID NO:24 corresponds to the predicted mature sequence of engineered phytase PHY-13868.

SEQ ID NO:25 corresponds to the predicted mature sequence of engineered phytase PHY-13883.

SEQ ID NO:26 corresponds to the predicted mature sequence of engineered phytase PHY-13885.

SEQ ID NO:27 corresponds to the predicted mature sequence of engineered phytase PHY-13936.

SEQ ID NO:28 corresponds to the predicted mature sequence of engineered phytase PHY-14004.

SEQ ID NO:29 corresponds to the predicted mature sequence of engineered phytase PHY-14215.

SEQ ID NO:30 corresponds to the predicted mature sequence of engineered phytase PHY-14256.

SEQ ID NO:31 corresponds to the predicted mature sequence of engineered phytase PHY-14277.

SEQ ID NO:32 corresponds to the predicted mature sequence of engineered phytase PHY-14473.

SEQ ID NO:33 corresponds to the predicted mature sequence of engineered phytase PHY-14614.

SEQ ID NO:34 corresponds to the predicted mature sequence of engineered phytase PHY-14804.

SEQ ID NO:35 corresponds to the predicted mature sequence of engineered phytase PHY-14945.

SEQ ID NO:36 corresponds to the predicted mature sequence of engineered phytase PHY-15459.